Widespread Elevational Occurrence of Antifungal Bacteria in Andean Amphibians Decimated by

14 Pages·2017·1.83 MB·English

by  Alessandro Catenazzi

fmicb-09-00465 March12,2018 Time:13:3 #1
ORIGINALRESEARCH
published:14March2018
doi:10.3389/fmicb.2018.00465
Widespread Elevational Occurrence
of Antifungal Bacteria in Andean
Amphibians Decimated by Disease:
A Complex Role for Skin Symbionts
in Defense Against Chytridiomycosis
AlessandroCatenazzi1,2*,SandraV.Flechas3,DavidBurkart2,NathanD.Hooven2,
JosephTownsend2 andVanceT.Vredenburg4
1DepartmentofBiologicalSciences,FloridaInternationalUniversity,Miami,FL,UnitedStates,2DepartmentofZoology,
SouthernIllinoisUniversity,Carbondale,IL,UnitedStates,3DepartmentofBiologicalSciences,UniversidaddeLosAndes,
Bogotá,Colombia,4DepartmentofBiology,SanFranciscoStateUniversity,SanFrancisco,CA,UnitedStates
Emerginginfectiousdiseaseisagrowingthreattoglobalhealth,andrecentdiscoveries
reveal that the microbiota dwelling on and within hosts can play an important
Editedby: role in health and disease. To understand the capacity of skin bacteria to protect
SpencerV.Nyholm,
amphibianhostsfromthefungaldiseasechytridiomycosiscausedbyBatrachochytrium
UniversityofConnecticut,
UnitedStates dendrobatidis (Bd), we isolated 192 bacterial morphotypes from the skin of 28
Reviewedby: host species of frogs (families Bufonidae, Centrolenidae, Hemiphractidae, Hylidae,
GraziellaVittoriaDiRenzo, Leptodactylidae,Strabomantidae,andTelmatobiidae)collectedfromtheeasternslopes
UniversityofCalifornia,
of the Peruvian Andes (540–3,865 m a.s.l.) in the Kosñipata Valley near Manu
SantaBarbara,UnitedStates
VijaykumarPatra, National Park, a site where we previously documented the collapse of montane frog
MedizinischeUniversitätGraz,Austria
communities following chytridiomycosis epizootics. We obtained isolates through agar
*Correspondence:
culture from skin swabs of wild frogs, and identified bacterial isolates by comparing
AlessandroCatenazzi
[email protected] 16S rRNA sequences against the GenBank database using BLAST. We identified 178
bacterial strains of 38 genera, including 59 bacterial species not previously reported
Specialtysection:
from any amphibian host. The most common bacterial isolates were species of
Thisarticlewassubmittedto
MicrobialSymbioses, Pseudomonas,Paenibacillus,Chryseobacterium,Comamonas,Sphingobacterium,and
asectionofthejournal Stenotrophomonas. We assayed the anti-fungal abilities of 133 bacterial isolates from
FrontiersinMicrobiology
26 frog species. To test whether cutaneous bacteria might inhibit growth of the fungal
Received:10November2017
pathogen, we used a local Bd strain isolated from the mouthparts of stream-dwelling
Accepted:28February2018
Published:14March2018 tadpoles (Hypsiboas gladiator, Hylidae). We quantified Bd-inhibition in vitro with co-
Citation: culture assays. We found 20 bacterial isolates that inhibited Bd growth, including three
Catenazzi A,Flechas SV,Burkart D,
isolates not previously known for such inhibitory abilities. Anti-Bd isolates occurred on
Hooven ND,Townsend Jand
Vredenburg VT(2018)Widespread aquatic and terrestrial breeding frogs across a wide range of elevations (560–3,695
ElevationalOccurrenceofAntifungal m a.s.l.). The inhibitory ability of anti-Bd isolates varied considerably. The proportion
BacteriainAndeanAmphibians
of anti-Bd isolates was lowest at mid-elevations (6%), where amphibian declines
DecimatedbyDisease:AComplex
RoleforSkinSymbiontsinDefense have been steepest, and among hosts that are highly susceptible to chytridiomycosis
AgainstChytridiomycosis.
(0–14%). Among non-susceptible species, two had the highest proportion of
Front.Microbiol.9:465.
doi:10.3389/fmicb.2018.00465 anti-Bd isolates (40 and 45%), but one common and non-susceptible species
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Catenazzietal. AntifungalBacteriainAndeanAmphibians
had a low proportion (13%). In conclusion, we show that anti-Bd bacteria are widely
distributedelevationallyandphylogeneticallyacrossfrogspeciesthathavepersistedina
regionwherechytridiomycosisemerged,causedadevastatingepizooticandcontinues
toinfectamphibians.
Keywords: 16S rRNA gene, amphibian declines, amphibian skin bacteria, antifungal bacteria,
elevationalgradient,montanediversitygradient,neotropical,tropicalAndes
INTRODUCTION amphibian communities (Bresciano et al., 2015; Hughey et al.,
2017).Aputativesourceofamphibianskinbacteria,soilbacterial
Emerging infectious disease is a growing threat to global communities are known to change with elevation and slope
health and is identified as a major factor involved in the aspect on mountains (Bryant et al., 2008). We hypothesize two
currentbiodiversitycrisis(Daszaketal.,1999).Amphibiansare main types of drivers affecting bacterial species composition at
considered one of the most threatened group of vertebrates on our study site, one related to environmental variation and the
earth(Stuartetal.,2004;WakeandVredenburg,2008;Catenazzi, other to host behavior. We hypothesized that the environment
2015),andtherecentlyemergeddiseasechytridiomycosis,caused (water, soil, leaf surface, etc.) is the main source of bacteria
by the fungal pathogen Batrachochytrium dendrobatidis (Bd), colonizing the skin of amphibians and that a species’ natural
has decimated species of amphibians in many parts of the history (elevational range, aquatic vs. terrestrial reproduction)
world (Berger et al., 1998; Skerratt et al., 2007; Kilpatrick influences its encounters with bacterial sources. At our study
et al., 2010). The microbiota that live on and within hosts sitethemaindeterminantofenvironmentalchangeiselevation,
can play an important role in health and disease (Harris whichiscorrelatedwithchangesintemperature,rainfall,oxygen
et al., 2009; Hanada et al., 2010; Hoyt et al., 2015; Ford availability, and UV radiation. Bacterial species richness in
and King, 2016). To understand the role of skin bacteria in three habitats (the phyllosphere, the mineral and the organic
protecting amphibians from chytridiomycosis, we document soil) does not vary consistently along the elevational gradient,
the distribution of amphibian skin bacteria with antifungal but bacterial community composition varies across these three
properties in host communities along the eastern slopes of the habitats, and within each habitat, across elevation (Fierer et al.,
tropicalAndeswhereamphibianpopulationshavecollapsedafter 2011; Nottingham et al., unpublished). Thus, it is possible that
Bdepizootics(Catenazzietal.,2011).Thisregionhasthehighest thespeciescompositionofbacterialcommunitiesonamphibian
amphibian species richness on Earth (Hutter et al., 2017), and skincouldvarywithelevation.
alongourelevationalgradientamphibianrichnesschangesfrom In addition to environmental factors, strains (or isolates;
more than 60 species in the Andean foothills to six species of thesetwotermsareinterchangeablethroughoutthemanuscript)
amphibians in the high-elevation grasslands (Catenazzi et al., colonizing the amphibian skin from the environmental pool
2013).Inourpreviouswork(Burkartetal.,2017),weshowedthat may be filtered by host identity and behavior (McKenzie
culturable skin bacteria inhibiting Bd growth (henceforth, anti- et al., 2012; Kueneman et al., 2014). Possible host-associated
Bd isolates) is linked to resistance to chytridiomycosis in high- factors include skin morphology, the type and diversity of
elevation frogs. Here, we extend this approach to the broader antimicrobial peptides secreted by skin, microhabitat use (e.g.,
elevationalgradient,includingelevationswherespeciesdeclines use of microhabitats in close contact with soil; Figure 1),
havebeensteepestfrom1,250to1,750ma.s.l.(Catenazzietal., reproductive mode (e.g., requiring extended time in water),
2011),andrelatethepresenceofanti-Bdbacteriaonfrogstohost and hydric and thermoregulatory behaviors (Rollins-Smith and
susceptibilitytochytridiomycosis(Catenazzietal.,2017). Woodhams, 2011; Bletz et al., 2017). Amphibians differ widely
Amphibian skin provides an excellent environment for the in their reproductive modes (Wells, 2007), which are linked to
growth of a wide variety of microorganisms (Kueneman et al., host affinity with aquatic environments. Adults of species with
2014;Bletzetal.,2017;Prado-Irwinetal.,2017).Thesemicrobial aquaticeggsand/ortadpolesoftencongregateinoraroundwater
communitiesarefunctionallyimportantforthehost,forexample bodies to attract mates, mate, lay eggs, and attend progeny (for
inthedefenseagainstpathogens,suchasBd(BettinandGreven, species with parental care). In contrast, adults of species that
1986; Harris et al., 2006; Belden and Harris, 2007; Culp et al., layeggsonlandmayrarelyapproachwaterbodies,andmaybe
2007; Woodhams et al., 2007b; Lauer et al., 2008; Becker and morehomogeneouslydistributedontheforestlitterorarboreal
Harris, 2010; Flechas et al., 2012). Although many amphibian vegetationfarfrompondsorstreams.
species have succumbed to epizootics of chytridiomycosis, Traditional,culture-basedmethodstostudybacterialdiversity
others persist despite infection. Resistance and tolerance to are limited in that only a fraction of the taxa present can be
chytridiomycosishasbeenattributedtothepresenceofbeneficial isolated and cultured. The use of next-generation sequencing
bacteria in the skin, which can delay or inhibit the growth of has improved our understanding of microbial community
the pathogen allowing host survival (Harris et al., 2006, 2009; composition (Qin et al., 2010; Fierer et al., 2011; Russo et al.,
Woodhamsetal.,2007a). 2012).Nonetheless,culture-basedmethodscanprovideaccurate
Amphibian skin bacteria are likely to vary across hosts information on the functions of a specific bacterium (Becker
and elevation, as seen in recent studies of other Neotropical et al., 2015b), and relevance of these functions to its symbiotic
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considering how little is known about amphibian skin bacteria
in the tropical Andes (Bresciano et al., 2015; Burkart et al.,
2017), the overarching goal of this study is to examine the
role of skin bacteria, and their anti-Bd abilities, in protecting
amphibian hosts from disease at our study site. Here, we aim
to(1)documentspeciescompositionofbacterialisolatesacross
elevation,hostidentityandbehavior,(2)determinetheinhibitory
abilityofbacterialisolatesagainstthelocalstrainofBd,and(3)
comparethedistributionofanti-Bdisolateswithrespecttohost
susceptibility,hostbehaviorandelevation.Thewideelevational
range,largenumberofhosts(28totalspecies),highsusceptibility
(six susceptible out of nine species with known status), and
diversityofhostreproductivemodes(spanningfromaquaticeggs
andtadpolestoterrestrialviviparousembryos),provideanideal
opportunity to investigate the contribution of skin bacteria to
hostdefenseagainstchytridiomycosis.
MATERIALS AND METHODS
Study System
We worked along the Paucartambo–Pillcopata road traversing
FIGURE1|Representativeamphibianhostsandtheirhabitatssampledfor the Kosñipata Valley and bordering Manu National Park and
thisstudy:(A,B)Hypsiboasgladiatorisnon-susceptibletochytridiomycosis its buffer zone in southern Peru (Catenazzi et al., 2011, 2014).
andlaysaquaticeggsinstreamsidebasinsalongmontanestreamsinthe This road connects the high-Andean grasslands (puna) of the
cloudforest;(C,D)Psychrophrynellausurpatorisnon-susceptibleandlays
Cordillera de Paucartambo at the mountain pass of Acjanaco
terrestrialeggsthatundergodirectdevelopmentundermossesinthe
(3,400ma.s.l.)totheAndeanfoothills(Pillcopata,540ma.s.l.)
high-Andeangrassland;(E,F)Telmatobiusmarmoratusishighlysusceptibleto
chytridiomycosisandlaysaquaticeggsinsmall,high-Andeanstreams. andAmazonianlowlandrainforestoftheupperMadredeDios
PhotographsbyA.Catenazzi. watershed. Owing to its wide elevational span and diversity of
ecosystems,ManuNPprotectsthehighestnumberofamphibian
speciesofanyprotectedarea(Catenazzietal.,2013).Wesampled
organism. Many studies have tested the antifungal properties 131 frogs belonging to 28 species in seven families distributed
of bacteria harbored on the skin of different amphibian species from560to3,865ma.s.l.inthesubmontaneandmontaneforests,
(Harris et al., 2006; Woodhams et al., 2007b; Lauer et al., montane scrub, and puna of the Kosñipata Valley (Table 1)
2008; Becker and Harris, 2010) and demonstrated that either during the dry season (14 June–22 August 2012). Furthermore,
a high proportion of anti-Bd bacteria, or bacteria with strong we include Telmatobius marmoratus (Telmatobiidae) obtained
antifungal capacities, might explain the survival of some host from the drier puna region surrounding the city of Cusco in
species (Flechas et al., 2012; Burkart et al., 2017). Bacterial JuneandJuly2012.ThisspeciesdoesnotoccurintheKosñipata
species belonging to the genera Pseudomonas, Serratia, and Valley,butisdistributedonthewesternsideoftheCordillerade
Janthinobacteriumareamongthosethathavedemonstratedgreat PaucartamboatshortdistancefromtheAcjanacomountainpass,
abilities to inhibit Bd growth (Becker et al., 2015a,b; Bresciano andisknownforhavinghighBdprevalenceandsusceptibilityto
et al., 2015), but there is a wide variety of other genera with Bd(Catenazzietal.,2010,2017;Warneetal.,2016).
similar abilities (Woodhams et al., 2015). Thus, there is a need
Ethics Statement
to combine culture-based methods with molecular analyses to
exploreamphibianskinbacterialcommunitiesinrelationtoBd This work has been approved by the Animal Care
resistanceandtolerance(Laueretal.,2008;Flechasetal.,2012; and Use Committees of San Francisco State University
Muletz-Wolzetal.,2017). (Protocol#A12-07) and Southern Illinois University (Protocol
We explored amphibian skin bacteria in host communities #13-027), and by the Peruvian Ministry of Agriculture. The
that experienced a collapse in species richness following Asociación para la Conservación de la Cuenca Amazónica
epizootics of chytridiomycosis (Catenazzi et al., 2011, 2014). authorizedworkatitsWayqechaBiologicalStation.
We previously discovered that the impact of Bd on amphibian
Disease Prevalence
communities was strongest among stream-breeding species
(Catenazzietal.,2011),andinamphibiancommunitiesatmid- Weestimateddiseaseprevalencebyswabbing543frogscaptured
elevations where Bd growth is optimal (Catenazzi et al., 2014). from 540 to 3,865 m a.s.l. with sterile synthetic rayon swabs
ManyamphibianspersistdespitethecontinuouspresenceofBd, (MW113, Medical Wire and Equipment, England). We gently
butseveralofthesepersistingspeciescontinuetobesusceptible stroked the sterile swab across the skin of each frog a total of
tochytridiomycosis(Catenazzietal.,2017).Inthiscontext,and 30 times: five strokes on each side of the abdominal midline,
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TABLE1|Speciesofanuransandelevationssampledforskinbacteria.
Hostspecies(samplesize) #tes/isl Elevation(m) Reproduction[modeinWells(2007)]
Bufonidae
Rhinellamanu(3) 0/4 1,920–2,100 Terrestrial,directdevelopment(23)
Centrolenidae
Hyalinobatrachiumbergeri(3) 6/6 1,030–1,920 Epiphyllouseggs,lotictadpoles(25)
Hemiphractidae
Gastrothecaantoniiochoai(2) 2/2 2,915 Marsupial,directdevelopment(35)
Gastrothecaexcubitor(11) 14/15 3,340–3,695 Marsupial,directdevelopment(35)
Gastrothecanebulanastes(6) 9/9 2,790–2,920 Marsupial,directdevelopment(35)
Gastrothecatestudinea(1) 2/2 2,100 Marsupial,directdevelopment(35)
Hylidae
Dendropsophusrhodopeplus(2) 2/2 560 Lenticeggsandtadpoles(1)
Hypsiboasgladiator(9) 10/12 1,350–1,450 Streamsidebasineggsandtadpoles(4)
Hypsiboaspunctatus(3) 2/3 560 Streamsidebasineggsandtadpoles(4)
Osteocephalusmimeticus(1) 1/1 1,100 Streamsidebasineggsandtadpoles(4)
Scinaxruber(4) 0/4 560–1,410 Lenticeggsandtadpoles(1)
Leptodactylidae
Adenomeraandreae(1) 1/1 540 Terrestrialfoamnest,non-feedingtadpoles(32)
Engystomopsfreibergi(1) 1/1 540 Aquaticfoamnestandlentictadpoles(11)
Strabomantidae
Bryophrynecophites(1) 2/2 3,865 Terrestrial,directdevelopment(23)
Noblellapygmaea(1) 1/2 2,970 Terrestrial,directdevelopment(23)
Oreobatesamarakaeri(1) 1/2 560 Terrestrial,directdevelopment(23)
Pristimantiscf.cruciocularis(1) 1/1 930 Terrestrial,directdevelopment(23)
Pristimantisdanae(10) 14/15 1,410–2,000 Terrestrial,directdevelopment(23)
Pristimantiscf.diadematus(1) 1/1 1,065 Terrestrial,directdevelopment(23)
Pristimantislindae(1) 1/1 1,920 Terrestrial,directdevelopment(23)
Pristimantispharangobates(15) 3/28 1,920–2,790 Terrestrial,directdevelopment(23)
Pristimantispluvialis(1) 2/2 930 Terrestrial,directdevelopment(23)
Pristimantiscf.platydactylus(6) 8/10 1,920–1,990 Terrestrial,directdevelopment(23)
Pristimantissalaputium(10) 14/19 1,350–1,450 Terrestrial,directdevelopment(23)
Pristimantiscf.toftae(7) 3/10 1,350–1,450 Terrestrial,directdevelopment(23)
Pristimantissp.(1) 1/1 2,350 Terrestrial,directdevelopment(23)
Psychophrynellausurpator(13) 15/17 2,950–2,975 Terrestrial,directdevelopment(23)
Telmatobiidae
Telmatobiusmarmoratus(15) 16/19 3,400 Streamsidebasineggsandtadpoles(4)
Total(samplesize:131) 133/192 540–3,865
#tes=numberofmorphotypestestedforBdinhibition(duplicatesconsideredassinglemorphotype);#isl=numberofmorphotypesisolatedfromhostspecies(after
discardingduplicates).Notethatnotallmorphotypescouldbesequenced.
five strokes on the inner thighs of each hind leg, and five Bayesian inference with Jeffrey’s non-informative priors with
strokes on the foot webbing of each hind leg (Catenazzi et al., the‘binom’packageinR(Dorai-Raj,2014).Bdprevalencedata
2011,2017).WeextractedDNAfromswabswithPrepmanUltra are available online at the Amphibian Disease database1 at the
(Life Technologies, Carlsbad, CA, United States). Each single URL:https://n2t.net/ark:/21547/AXY2.
swab extract was amplified once following a standard, probe-
based quantitative Polymerase Chain Reaction protocol (Boyle Bacterial and Bd Isolation
et al., 2004; Hyatt et al., 2007) using a 7300 Real-Time PCR We obtained bacteria directly from skin swabs of frogs. We
System (Life Technologies, Carlsbad, CA, United States), as handledeachfrogwithnewnitrileglovesandrinsedthemwith
reported in Catenazzi et al. (2017). This qPCR assay estimates distilled water prior to swabbing in order to remove transient
Bdgenomicequivalents(GEs)ineachsample,andweconverted cutaneousbacteria(Laueretal.,2007).Wesampledbacteriaby
GE values to provide “zoospore equivalents” on each frog by running a sterile MW113 swab (not the same swab used for
usinggenomicstandardsofknownzoosporeconcentrations.We determining Bd infection) on the frog’s left and right sides and
considered frogs to be infected if zoospore equivalents > 0, ventral surfaces, hindlimbs, and interdigital membranes for a
andnon-infectedifzoosporeequivalents=0.WecalculatedBd
prevalence(proportionofswabbedfrogsinfectedwithBd)using 1https://amphibiandisease.org/
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total of 50 strokes (Flechas et al., 2012). We then streaked the interface we used2 allows computation of multiple sequence
swab on a petri dish of nutrient agar and incubated the dish at alignment for large datasets (i.e., thousands of sequences), and
room temperature (14–18◦C) until observing bacterial growth calculates genetic distances for the aligned sequences. We used
(4–6 days). We defined bacterial morphotypes according to 1% 16S genetic distance as our criterion for delimitation of
macroscopiccharacteristics(color,form,elevation,andmargin), operativetaxonomicunits(OTUs).
and transferred each to fresh nutritive agar plates until pure
cultures were obtained. We transported each isolate to the Bd Growth Inhibition Assays
laboratory for Bd growth inhibition assays and identification
We used agar plate co-cultures to assay the ability of bacterial
through sequencing of the 16S rRNA gene (Weisburg et al.,
strainstoinhibitBdgrowth.WeflushedplateswithaBd-enriched
1991).
broth to produce a homogeneous distribution of Bd colonies.
We isolated a local strain of Bd from the mouthparts of
Then we streaked a line of the bacterial isolate on one side of
HypsiboasgladiatortadpolescollectedincreeksoftheKosñipata
theplate,paralleltoasecondlineofabacterialstrainknownto
Valley at 1,350 m a.s.l. We examined the mouthparts for signs lackanti-Bdproperties(EscherichiacolistrainDH5α)servingas
of Bd infection, such as depigmentation and missing tooth
negativecontrol.Weassayedbacterialstrainsintriplicate.After3
rows (Fellers et al., 2001; Marantelli et al., 2004; Rachowicz daysincubatedat23◦C,wevisuallyinspectedplatesandclassified
and Vredenburg, 2004). We euthanized individuals with signs
the bacterial strains as either non-inhibitory (no zone of Bd
of disease by decapitation, their mouthparts dissected and cut
growthinhibitionpresent)orpotentiallyinhibitory(clearzoneof
into approximately 2 mm × 2 mm squares. We cleaned each
Bdgrowthinhibitionpresent).Weexaminedplatesafter3days
piece of mouthpart in water-based agar with antibiotics in
because that time is when maximum Bd zoospore production
order to remove bacteria, yeast, and fungal spores. We then
was observed during our protocol optimization procedures.
transferredthecleanmouthpartstoafreshplatewithTGhmedia
Furthermore, many bacteria grew quickly, and the third day
(10 g tryptone, 10 g agar, 4 g gelatin hydrolysate, 1,000 mL
wasoptimalforquantifyingBdinhibitionandcomparingvalues
distilled water). We transferred Bd colonies to fresh plates
amongbacterialisolates.
as soon as they appeared, and these were transported to the
We quantified the relative distance from the streak of the
laboratoryandmaintainedincultureonTGhagarat23◦C.We
querybacteriumtowhere50%ofmaximumBdgrowthoccurred,
cryopreservedtheBdstrainfollowingstandardprocedure(Boyle
andusedthisvaluetocompareeachisolate’sstrengthatinhibiting
etal.,2003).
Bd (Burkart et al., 2017). Isolates with stronger inhibitory
abilitiesdepressedBdgrowthoverlongerdistances.Werecorded
Bacterial DNA Extraction, Sequencing, Bd growth by measuring the gray values of standardized
photographs of the petri dishes using ImageJ software (Flechas
and Identification
etal.,2012;Burkartetal.,2017).Grayvalues(hereafterreferred
We swabbed agar plates with pure cultures of each bacterial
to as “growth”) are measurements of the intensity of light in
strain, then extracted DNA from these swabs using Prepman
a pixel of a black and white image, and by averaging among
Ultra, and amplified DNA by PCR with GoTaq Green Master
pixelwidecolumnsacrosstheareabetweenthequerybacterium
Mix (Promega Corporation, Madison, WI, United States) and
andnegativecontrol,wequantifiedgrowthoverminutedistance
16S primers 27F and 1492R (Lane, 1991). After verifying PCR
increments from the query. Distances between the query and
products by gel electrophoresis, we sequenced each isolate’s
the negative control varied slightly between petri dishes, so we
DNA (MCLAB, San Francisco, CA, United States). We aligned
expressed them as percentages of the total length (i.e., relative
sequences(onlythosewith>60%highqualitybasepairs)using
distance)tofacilitatecomparativeanalysis.Weexpressedgrowth
Geneious v8.1.9 (Biomatters Limited, Auckland, New Zealand),
as percentage of the maximum growth recorded to control for
andcomparedconsensussequencestomicrobialDNAsequences
anyvariationbetweenpetridishes.Foreachreplicate,weplotted
ontheNCBIdatabase(usingBLASTdefaultparameters).Isolates
valuesonagraphusingMicrosoftExcel(MicrosoftCorporation,
thatwere>99%similarwereconsideredamatch(Burkartetal.,
Redmond, WA, United States), and determined the distance at
2017).
which50%growthoccurredusingthefunctionofalinefittothe
We could not assay 50 of our bacterial isolates for Bd
graphedvalues(Burkartetal.,2017).Wereportaveragerelative
inhibition (see below). Therefore, we attempted to infer
distanceandstandarderrorcalculatedacrossthethreereplicates
inhibitory status for these isolates from a published dataset
ofeachisolate.
(Woodhams et al., 2015). We also included in this comparison
Fourbacterialstrainswereassayedtwice(becausestrainshad
our assayed isolates to examine consistency of inhibitory status
not been sequenced prior to assays, and thus morphologically
across studies. In order to compare our sequenced isolates
variable strains were thought to be distinct following visual
with the published literature, we retained 159 sequences after
examination), three strains were non-inhibitory across the two
discarding five duplicate and three triplicate sequences and 19
trials(Paenibacillussp.2;Pseudomonassp.24;Chryseobacterium
lowqualityreads.These159sequences(seeGenBankaccession
lactis); whereas Pseudomonas sp. 2 was inhibitory in the first
codes in Supplementary Table S1) were added to the dataset of
trial (average inhibition strength of 67%), and non-inhibitory
amphibian skin bacteria of Woodhams et al. (2015), and the
combined list of 2102 strains was aligned using MAFFT v7.0
usingthedefaultoptionFFT-NS-2(Katohetal.,2017).Theonline 2https://mafft.cbrc.jp/alignment/server/
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in the second trial. These four strains were considered to be variablescanbeused.Becausetherewaslittleoverlapofbacterial
non-inhibitoryforouranalyses. speciesandgeneraamongamphibianhosts,weconsidereddata
ondistributionofphylaforNMDS.Weperformedanalysesinthe
Host Susceptibility Data and Statistical
Rpackagevegan(Oksanenetal.,2015)byusingthe“metaMDS”
Analyses
function for NMDS, the “rankindex” function to determine
We used data from infection experiments conducted as part of the best method for calculating the distance matrix from our
anotherstudy(Catenazzietal.,2017)torelatehostsusceptibility data, and the “adonis” function to perform analysis of variance
to proportion and strength of anti-Bd bacterial strains. Frogs (permutation test with pseudo-F ratios) using the distance
of eight species sampled in this study (Gastrotheca excubitor, matrices and five categorical variables (elevation, reproductive
G. nebulanastes, Hypsiboas gladiator, Pristimantis danae, P. mode,hostsamplesize,andhostsusceptibility).Forthepurpose
cf. platydactylus, P. pharangobates, P. toftae, Psychrophrynella of these analyses of variance, we conflated reproductive modes
usurpator) were exposed to highly infected individuals of into two categories (terrestrial eggs and aquatic eggs), and
Telmatobius marmoratus. Following infection, we compared performedtheanalysisforhostsusceptibilityonareduceddataset
survival between groups of Bd-exposed and non-exposed (information on susceptibility only available for nine of the 28
(control)individuals(durationofexperimentsvariedbyspecies). sampledamphibianspecies).Wereportaverages±SE.
Frogs were cleared of Bd infection before the experiments by
immersion in a 1% itraconazole solution for 5 min a day for
RESULTS
seven consecutive days. We used Cox’s proportional hazards
modelwithcensoringtoassesstheriskofdyingforBd-exposed
Composition of Cutaneous Bacteria
frogs (Catenazzi et al., 2017). Here, we use the predicted days
to death from Cox’s proportional hazards models for each Weisolated199bacterialmorphotypesfromtheskinof131frogs
species as reported in Catenazzi et al. (2017), and relate this distributedacross28speciesandsevenanuranfamilies,andfrom
value to the proportion and inhibitory strength of anti-Bd 540 to 3,865 m a.s.l. (Table 1). We were able to sequence 185
strains. We used analysis of variance to determine whether of these 199 morphotypes, and among these 185 sequences, we
the proportion of anti-Bd isolates predicts the number of days identifiedfiveduplicatesandonetriplicate,reducingthenumber
to death. For descriptive purposes, we consider host species of bacterial isolates to 178 OTUs (Supplementary TableS1).
to be Bd-susceptible whenever our survival analysis models in These OTUs were distributed across four bacterial phyla:
Catenazzietal.(2017)producedanestimatefornumberofdays Proteobacteria,Actinobacteria,Firmicutes,andBacteroidetes.
to death. We also include data of survival for T. marmoratus The number of OTUs varied with host sample size
(not included in Catenazzi et al., 2017) comparing survival (F1,26 = 366.3, p < 0.001), and after taking into account this
between infected and treated frogs (immersion in itraconazole sampling effect, there was no change in the average number
baths). of OTUs per host across elevation (F1,5 < 0.01, p = 0.977).
We examined patterns of bacterial composition at two There were some changes in the relative proportion of phyla
taxonomiclevels:strain(OTU)andphylum.Weusedanalysisof acrosselevation(Figure2).Atallelevations,isolatedskinbacteria
variancetotestthesignificanceoflinearregressionrelatingfrog were dominated by Proteobacteria, followed by Firmicutes,
samplesizewithnumberofOTUs.Inordertoexploretheeffect Bacteroidetes, and Actinobacteria, but the relative proportion
of elevation on number of OTUs, skin bacterial composition of Bacteroidetes peaked at 1,000–1,499 m a.s.l., and that of
andproportionofanti-Bdisolates,weusedanalysisofvariance Firmicutesat2,500–2,999ma.s.l.
and pooled samples into seven 500 m elevational classes (540– Analyses of variance (permutation test) showed that host
999m,1,000–1,499m,etc.upto3,500–3,865ma.s.l.).Wealso samplesize(F1,23=51.29;p=0.001)andelevation(F1,23=4.13;
fitted a polynomial, quadratic curve to model the relationship p = 0.034) significantly affected phyla community composition
betweenelevationandtheproportionofanti-Bdisolates.Inorder across amphibian hosts (Supplementary Figure S1), but there
to explore the effect of host reproductive behavior, we used a werenoeffectsofegglayingsite(F1,23 =0.67;p=0.521),and
χ2 contingency table test with the categories of reproductive hostsusceptibility(F1,7=−0.08;p=0.978).
mode proposed by Wells (2007), arranged along a continuum
Distribution of Anti-Bd Bacteria and Host
from aquatic eggs and tadpoles, to direct development on
land, and viviparity (marsupial frogs), as listed in Table 1. For Susceptibility to Chytridiomycosis
dimensionalanalysesofskinbacterialcommunitiesweusednon- We assayed 137 morphotypes, belonging to 133 isolates (four
metric multidimensional scaling (NMDS). NMDS produces an morphotypes were assayed twice), sampled from 26 species
ordination,i.e.,conflatesinformationfrommultipledimensions of frogs. Six of the 133 strains could not be sequenced
into a few dimensions based on a distance or dissimilarity (“undeterminedbacteria”;SupplementaryTableS1),fiveofwhich
matrix. NMDS is usually considered as a more robust and wereinhibitory.Therefore,ourdatasetofassayedstrainsincludes
flexibletechniquethanotherordinationtechniquesbecausethe 126 OTUs identified through sequencing of 16S rRNA, and six
originaldistancesarereplacedwithranks(Minchin,1987).While unidentifiedisolates.
the information on distance magnitude is thus lost, the rank Wefound21anti-Bdbacterialisolatesinninehostspeciesof
based approach is more robust for data lacking an identifiable sixgenera(Dendropsophus,Gastrotheca,Hypsiboas,Pristimantis,
distribution. Furthermore, mixed quantitative and qualitative Psychrophrynella, and Telmatobius; Table 2), with only two
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species, G. excubitor and H. gladiator, having more than four
anti-Bd isolates each. These hosts and their associated anti-
Bd bacteria live at elevations from 560 to 3,695 m a.s.l., in
habitats that span from foothill tropical rainforest to cloud
forest and high-Andean grassland, and hosts that lay eggs in
ponds(Dendropsophus),streams(Hypsiboas,Telmatobius),moist
terrestrial environments (Pristimantis, Psychrophrynella), and
thatretaineggsinspecializeddorsalpouches(Gastrotheca).
The proportion of anti-Bd isolates ranged from 0 to 14% in
six host species that were susceptible to Bd, but there was no
relationshipbetweennumberofdaystodeathandproportionof
anti-Bdisolates(F1,4 =1.46,p=0.293;Figure3A).Amongthe
threenon-susceptiblehostspecies,G.excubitorandH.gladiator
had the largest proportions of anti-Bd isolates (40 and 45%,
respectively), but P. usurpator only had 13% anti-Bd isolates,
withintherangeofBd-susceptiblehosts.
ThestrengthofBdinhibitionaveraged55.0±2.6%acrossall
isolates,andrangedfrom32.9to78.0%(Table2andFigure3B).
Althoughdatapointsaretoofewtocomputestatisticalanalyses,
FIGURE2|Changeinthecompositionoffrogskinbacterialisolatesarranged
byphylaalongtheelevationalgradientintheKosñipataValleynearManu
TABLE2|Distributionandinhibitorystrength(asmeasuredbytherelative
NationalPark,SEPeru.
distancefromthestreakofthequerybacteriumtopointof50%ofmaxBdgrowth;
seeMaterialsandMethods)ofanti-Bdbacterialisolatesacrosshostspecies.
Hostspeciesandisolate Elevation(m) Distance SE theinhibitionstrengthsofanti-Bdisolatesweregenerallyhigher
innon-susceptiblehoststhantheywereinBd-susceptiblehosts,
Dendropsophusrhodopeplus
with the exception of P. usurpator. Only three of the six Bd-
Pseudomonasentomophila 560 78.04% 2.20%
susceptiblehostsareshowninFigure3B,becausetheotherthree
Serratiamarcescens 560 42.43% 14.97%
specieshadnoanti-Bdisolates.Hostsofunknownsusceptibility
Gastrothecaexcubitor
toBdhadanti-Bdisolateswithinhibitionstrengthsimilartothat
Pseudomonassp.2 3,340 67.21% 10.39%
ofnon-susceptiblespecies.
Pseudomonassp.34 3,340 61.44% 10.77%
The proportion of anti-Bd isolates varied with elevation
Pseudomonassp.18 3,340 58.80% 7.67%
Rahnellaaquatilis 3,695 50.25% 9.10% (F2,4 = 36.05, p = 0.0028; Figure 4), following a polynomial,
quadratic curve that reaches its lowest values at mid-elevations
Pseudomonassp.1 3,695 47.91% 1.91%
from1,500to2,000ma.s.l.,partiallyoverlappingwithelevations
Janthinobacteriumlividum 3,695 32.86% 3.08%
from 1,250 to 1,750 m a.s.l. where at least 35% of amphibian
Gastrothecanebulanastes
Pseudomonassp.25 2,850 38.44% N/A hostsbecameextinctduringtheBdepizootics(Catenazzietal.,
Hypsiboasgladiator 2011).Theseelevationalrangesalsopartiallyoverlappedwiththe
Undeterminedbacterium14 1,410 69.98% 6.85% 1,500–2,200ma.s.l.rangewhereprevalenceofBdinfectionwas
Undeterminedbacterium13 1,410 61.38% 1.76% highest during our sampling period in the dry season of 2012
Rahnellasp.2 1,410 56.37% 4.47% (SupplementaryTableS2).
Undeterminedbacterium1 1,450 49.69% 9.77% Anti-Bd isolates occurred across four types of reproductive
Sphingobacteriumfaecium 1,350 43.68% N/A modes (Figure 5): aquatic eggs and tadpoles in ponds, aquatic
Pristimantiscf.diadematus eggs and tadpoles in streamside basins, terrestrial breeding
Paenibacillussp.24 1,065 66.64% 1.29% with direct development, and marsupial brooding with direct
Pristimantisdanae development. There was no effect of reproductive mode on the
Pseudomonassp.36 1,920 58.94% 9.96% proportionofanti-Bdisolates(χ2 =10.372,df=6,p=0.110)
Paenibacillussp.3 2,000 45.73% 6.93% and,amongthefourreproductivemodeswithanti-Bdisolates,no
Pristimantissalaputium effectontheinhibitorystrengthofanti-Bdisolates(F3,46=0.68,
Undeterminedbacterium2 1,350 73.50% 10.48% p=0.571;Figure5).
Psychrophrynellausurpator
Undeterminedbacterium5 2,970 58.26% 9.31% Comparison With Published Literature
Pseudomonasazotoformans 2,975 51.11% 11.02% ThealignmentproducedthroughMAFFTv7.0generatedshorter
Telmatobiusmarmoratus genetic distances than those calculated for our isolates in
Pseudomonasfluorescens 3,400 41.77% 2.43% Geneious. Despite these shorter distances, 59 of our 159
Bacteriaarerankedaccordingtotheirinhibitoryabilities. sequenced isolates had no matches (our OTUs defined as those
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FIGURE3|(A)Variationinproportionofanti-BdbacterialisolatesinBd-susceptibleandnon-susceptibleamphibianhosts.Insusceptiblespecies,daystodeath
afterBdinfectionarecalculatedfromsurvivalanalysis(Catenazzietal.,2017).Numberoftestedbacterialmorphotypesreportedaboveeachdatapoint.(B)Average
strength(±SE)ofBdinhibitionofanti-Bdisolates(asmeasuredbytherelativedistancefromthestreakofthequerybacteriumtopointof50%ofmaxBdgrowth;
seeMaterialsandMethods)rankedbyinhibitorystrength(fromstrongesttoweakestinhibitor)inthreeBd-susceptiblehosts(inred),threenon-susceptiblehosts(in
blue),andthreehostsofunknownBdsusceptibility(i.e.,nottested,ingray).Bacterialstrainsvarybyfroghostspecies.
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FIGURE5|Variationinaveragestrength(±SE)ofBdinhibitionofanti-Bd
isolates(rankedfromstrongesttoweakestinhibitor)acrosshostreproductive
modes.
FIGURE4|Changeinproportionofanti-Bdbacterialstrainswithinphyla
(bars)andacrossallphyla(circles;numbersindicatehostsamplesize)along
theelevationalgradientintheKosñipataValleynearManuNationalPark,SE
attempt at inferring inhibitory status of bacterial isolates by
Peru.Diamondsindicatedisease(Bd)prevalencewithBayescredibleintervals
inamphibianhosts,andtheshadedareaindicateselevationsatwhichatleast matching their 16S sequences with sequences from previous
35%ofamphibianspecieshavedisappearedfollowingtheBdepizootics studieswasunsuccessful,suggestingthatOTUidentityisapoor
(Catenazzietal.,2011). predictorofinhibitionstrength.
Our findings acquire relevance in the context of our study
system, which consists of species-rich amphibian communities
having 16S genetic distance <1%) among the 1,944 previously that have been decimated by chytridiomycosis. This highly
published isolates listed in Woodhams et al. (2015). The virulent disease infects the skin and interferes with the organ’s
remaining100isolatesproduced445matcheswithisolatesinthe functions, eventually leading to death in many amphibian
publisheddataset,withsomeisolatesofPseudomonasgenerating species (Voyles et al., 2009; Kilpatrick et al., 2010). We have
upto100(Pseudomonasentomophila),81(Pseudomonassp.7), previouslydocumentedthecollapseofmontaneforestamphibian
and 74 matches (Pseudomonas sp. 2 and Pseudomonas sp. 24). communities at our study site (Catenazzi et al., 2011, 2014),
There was little consistency in inhibitory status among OTUs. following epizootics of chytridiomycosis that reached southern
None of the OTUs we identified as inhibitory were inhibitory Peruintheearly2000s(CatenazziandvonMay,2014).During
acrossallstudiessummarizedinthepublisheddataset.Likewise, these epizootics nearly 20 out of 60 species of amphibians
none of the OTUs we identified as non-inhibitory were non- were extirpated from the Kosñipata Valley, and many more
inhibitoryacrossallstudiessummarizedinthepublisheddataset. declinedinabundanceordisappearedfrompartoftheirprevious
Therefore,wedecidednottouseOTUasapredictorofinhibitory elevationalrange.Whilethehost/Bddynamicshaveshiftedfrom
statusfortheisolatesthatwecouldnotassayagainstBd. epizootic to enzootic (Catenazzi et al., 2017), and communities
ofsurvivingspeciesdonotseemtoundergofurtherdeclines,we
recently demonstrated that many surviving host taxa continue
DISCUSSION to be susceptible to chytridiomycosis (Catenazzi et al., 2017).
Therefore,host-specificfactors,orthedynamicsoftheenzootic
We show that bacterial isolates inhibiting the fungal disease host/Bd system, currently prevent Bd from causing widespread
chytridiomycosis are distributed across a wide range of host mortality and further population declines. With this study
environmentalconditions(temperatureandrainfall,astheyvary wehaveexploredonesuchfactor,hostskindefenseprovidedby
along elevation), host families and genera, host susceptibility symbioticbacteria.
status, and microhabitat associated with host reproductive Ourprimaryinterestwastoexamineanyrelationshipbetween
behavior. Our findings suggest anti-Bd isolates may play a distribution of anti-Bd isolates and host susceptibility in the
role in host defense against chytridiomycosis, maintenance of contextofamphibiancommunitiesravagedbychytridiomycosis.
host community composition, and the elevational pattern of We compared the elevational distribution of anti-Bd isolates
Bd infection prevalence. We found the highest proportion withtheelevationalrangecorrespondingtothesharpestdeclines
and inhibition strengths of anti-Bd isolates in two non- in amphibian diversity (where at least 35% of frog species
susceptible species, while Bd-susceptible species had low have been extirpated), and with elevational variation in Bd
proportions (<15%) of weakly inhibitory bacterial isolates. infectionprevalence.Differenceinelevationalresolutionamong
Intriguingly, we also recorded low proportion, and weak these datasets prevented more rigorous statistical analyses;
inhibitory strength in a common, non-susceptible species. Our data on proportion of declining amphibian species and Bd
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prevalenceareavailablefor100melevationalclasses(Catenazzi mid-elevations.BdprevalenceandinfectionloadsinP.usurpator
et al., 2011), whereas data on anti-Bd isolates were pooled are generally lower than in sympatric frog species (Catenazzi
into 500 m elevational classes to minimize differences in et al., 2011, 2017), suggesting that this species might avoid
sample size among classes. Assuming the observed patterns are infection, or that its skin might excel at preventing zoospore
representative, our data suggest that the lowest proportion of penetration,orthatitsadaptiveimmunesystemismoreeffective
anti-Bd isolates overlap with regions of high Bd prevalence atresistingchytridiomycosis(SavageandZamudio,2011).This
and high host extirpation, which creates a conundrum. One frogbreedsterrestriallyanddoesnotuseaquatichabitatswhere
would expect the opposite pattern to occur if Bd was selecting Bdzoosporesoccurathighestdensities,nordoesitcongregatefor
for host resistance. If symbiotic bacteria were contributing reproduction, thus limiting potential for Bd transmission from
to skin defenses, then hosts that survived Bd epizootics in both environmental reservoirs and highly infected individuals.
disease-ravaged communities should have greater proportions Behavioraleffectsandindependencefromwaterbodiesmayalso
of anti-Bd bacteria than survivors in communities that had explain why other terrestrial-breeding species, such as P. toftae
not been strongly diminished by disease. Another possibility whichoccursatmid-elevationinregionsofhighBdprevalence,
is that most frogs at mid-elevations lacked abundant beneficial cancontinuetopersistwithoutprotectionfromanti-Bdbacteria.
symbionts, explaining why so many species were extirpated Comparisons of our strains with a published dataset, after
during epizootics, and that surviving species continue to lack alignment of 16S sequences and using 1% genetic distance as
symbionts, but are able to persist for other reasons. Other criterion for OTU delimitation, revealed that OTU identity
skin defenses, such as antimicrobial peptides, play important is a poor predictor of inhibition status and strength, to the
roles in inhibiting Bd growth and delaying the lethal effects of extent that it thwarted our attempt to determine inhibition
chytridiomycosis(Rollins-Smithetal.,2002;MelzerandBishop, strength for isolates that we could not assay against Bd.
2010;Holdenetal.,2015).Moreover,diminishedfrogdensityand Although bacterial genera such as Jantinobacterium, Serratia,
lower number of host species following epizootic outbreaks, as Stenotrophomonas,Aeromonas,andPseudomonasarefrequently
wellasthetransitionofthehost/Bddynamicsfromepizooticto associated with anti-Bd capacity (Harris et al., 2009; Becker
enzooticlikelyreducesratesoftransmissionandtheavailability et al., 2015b; Woodhams et al., 2015), emergent traits of
ofenvironmentaldiseasereservoirs(Briggsetal.,2010),allowing bacterial communities, including community structure and
hostspeciestopersistandevenrecover(Knappetal.,2016). species richness (Woodhams et al., 2014; Becker et al., 2015a;
An alternative hypothesis to the conundrum posed by the Piovia-Scott et al., 2017), might be more predictive of host
U-shapedcurveinelevationaldistributionofanti-Bdisolatesis resistance to chytridiomycosis than data from co-culture assay
thathighprevalenceofBd,andthushighprobabilityofinfection testing single bacterial isolates. These findings are important
in hosts, is disrupting skin bacterial communities (Jani et al., to inform probiotic treatments aimed at mitigating the effects
2017), and disproportionally affecting anti-Bd bacteria relative of chytridiomycosis on amphibian hosts (Bletz et al., 2013;
to co-occurring bacteria. This alternative hypothesis would Kuenemanetal.,2016).
again imply that other factors, unrelated to skin symbionts, are Our investigation, which was performed by using culturable
preventinghighBdvirulenceinthesehosts.Continuousexposure bacteria,mayprovidealimitedrepresentationofoverallbacterial
toBdinregionsofhighprevalence,forexample,couldstrengthen diversity yet has the advantage of producing isolates that can
acquired immunity in hosts (McMahon et al., 2014), although be tested for anti-Bd capacity (Becker et al., 2015b). Previous
ourexperimentaldata(Catenazzietal.,2017)andfindingsfrom work has demonstrated that most dominant skin bacteria can
otherstudiesshowlimitedsupportforimmunoprotectiveeffects be isolated and cultured (Walke et al., 2015), so the undetected
inmanyspeciesofamphibians(SticeandBriggs,2010;Cashins bacteria may not constitute an extensive group. Furthermore,
etal.,2013;Hudsonetal.,2016;Geigeretal.,2017). culturable isolates were needed to determine isolate-specific
Support for a role of symbiotic bacteria in frog skin inhibition strength against the local strain of Bd. These strain-
defense is provided through our comparison of the inhibitory specific inhibition data were especially informative because we
strength and proportion of anti-Bd isolates in hosts that were previously determined host susceptibility to the same local Bd
experimentally infected with Bd. With the exception of one strain in situ (Catenazzi et al., 2017), and because inhibitory
common and non-declining frog species (P. usurpator), we capacity of bacteria appears to vary when different Bd strains
found that non-susceptible species had the highest proportion are assayed (Antwis et al., 2015). By sampling bacteria directly
of strongly inhibitory anti-Bd isolates, whereas Bd-susceptible from wild frogs, testing them against a local strain of Bd, and
species had a low proportion of weakly inhibitory anti-Bd relatingourfindingstoknownpatternsofdiseaseprevalenceand
isolates.Wepreviouslyassociatedvariationinthehostresponse susceptibility, we aim to minimize external factors that would
to Bd infection in marsupial frogs to the presence of bacteria interferewithourinterpretationregardingtheroleofsymbiotic
with antifungal properties (Burkart et al., 2017). We confirmed bacteriaasalineofdefenseagainstfungaldisease.
this association along the broader elevational gradient and Theenvironmenthasbeenhypothesizedasthemainsourceof
across nine frog species from four families. For the case of symbioticbacteriainfrogs(Muletzetal.,2012),althoughthereis
P. usurpator, a species that persisted and even increased in evidencethatthecoreofhostbacterialcommunitiesmayoccur
abundancedespitethepresenceofBd(Catenazzietal.,2017),the independently of environment (Wiggins et al., 2011; Loudon
proportion of anti-Bd bacteria is very low and similar to what etal.,2014).Verticaltransmissionofbacteriatooffspringhasalso
we found for Bd-susceptible species, and for species found at been suggested as an alternative route for species with parental
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